The type 1 parathyroid hormone receptor (PTH1R), a member of G-protein coupled receptors (GPCRs), mediates PTH actions to maintain bone mineral homeostasis. Osteoblast dysfunction leading to bone loss is thought to be a key mechanism in osteoporosis. Intermittent administration of PTH (1-34) stimulates bone formation in patients and in experimental animals. Continuous infusion of PTH (1-34), which mimics pathological changes in clinical hyperparathyroidism, causes bone resorption. Patient with end-stage renal disease develops a systemic disorder of mineral and bone metabolism, such as renal osteodystrophy. PTH1R down-regulation and skeletal resistance to PTH not only reduce the therapeutic effect of PTH treatment for osteoporosis, but also occur in diseases including secondary hyperparathyroidism and renal osteodystrophy. To protect against the PTH1R down-regulation is a complementary strategy that may be useful for treatment of these diseases. Although the signaling pathways of PTH receptor are reasonably well understood, the mechanism of regulation of PTH1R functions remains to be characterized. Postsynaptic density 95/discs large/zona occludens (PDZ) scaffolding proteins comprise a key class of GPCRs-interacting proteins that can strongly influence signaling and trafficking of GPCRs. We identified two novel PTH1R associating proteins by using a newly developed proteomic array of distinct PDZ domains and coimmunoprecipitation experiments. These PDZ scaffolding proteins, MAGI-3 and spinophilin, are endogenously expressed in osteoblasts. Both MAGI-3 and spinophilin increase PTH-induced cAMP formation in osteoblasts. Based on these observations, we hypothesize that MAGI-3 and spinophilin modulate PTH1R functions and protect against receptor down-regulation in bone. Three specific aims are developed to test this hypothesis. Aim 1 will determine the effects of MAGI-3 and spinophilin on PTH1R signaling in osteoblasts. In Aim 2, we will identify whether MAGI-3 and spinophilin protect against PTH1R down-regulation in osteoblasts. Aim 3 will define the role of spinophilin on bone formation bone resorption in mice treated with intermittent PTH. Successful completion of the proposed studies will provide novel and important information on the regulation of PTH1R signaling, trafficking, and functions by MAGI-3 and spinophilin in bone. The knowledge gained from these studies will provide important insight into therapeutics for the treatment of osteoporosis and other diseases related to PTH1R down-regulation such as secondary hyperparathyroidism and renal osteodystrophy.